Fibronectin (FN) was first reported by Morrison et al. [Morrison, P. R. et al., J. Am. Chem. Soc., 70, 3103 (1948)] as one of plasma proteins in 1948. Being a multifunctional protein broadly distributed in various tissues and body fluids, this substance is known to be involved, as a cell adhesion factor, in a large variety of biological events such as the migration, differentiation, proliferation and canceration of cells [Sekiguchi, K.: Cell Engineering, 4 (6), 485-497 (1985)].
Meanwhile, FN as it is synthesized in the liver and occurring in the blood is known as plasma FN (pFN), and FN as it is detected on the cultured cell surface and culture medium is called cellular FN (cFN) [Sekiguchi et al., J. Biol. Chem., 260 (8) 5105-5114 (1985)]. It has been shown that these species of FN are subject to molecular diversity due to alternative splicing of the early gene transcription product. As the regions subject to such alternative splicing, there are three regions called EDA, EDB and IIIcs, and it is believed that a large number of molecular species occur according to varied combinations of expression of these regions. In pFN, the above-mentioned EDA and EDB regions have not been appreciably expressed. On the other hand, cFN is an FN with a high degree of expression of said EDA region. Peters J. H. et al. conjugated a peptide having 29 amino acids with keyhole limpet hemocyanin (KLH) to prepare an immunogen, constructed an anti-cFN polyclonal antibody specifically reactive to cFN having the EDA region, and using the antibody, demonstrated that the normal human blood contains traces of cFN and that the blood cFN level is markedly elevated in patients with collagen disease accompanied by vasculitis [Am. Rev. Respir. Dis., 138, 167-174 (1988); J. Lab. Clin. Med., 113 ( 5), 586-597 (1989)]. According to the above report of Peters et al., the physiological implications of increased blood cFN in patients with collagen disease accompanied by vasculitis are not definitely known but a correlation between the stage or severity of disease and the blood concentration of cFN is suggested. The investigation made by the inventors of the present invention using the anti-cFN monoclonal antibody which is to be described hereinafter revealed that whereas the plasma concentration of cFN in healthy humans is 1 to 2 .mu.g/ml, it is 4 to 8 .mu.g/ml in patients with various diseases and as high as 10 and odd .mu.g/ml in seriously ill patients. On the other hand, pFN is invariably within the normal range of 270 to 400 .mu.g/ml, irrespective of subjects.
Therefore, it is a subject of intriguing interest to investigate how the course of disease is modified by selective elimination of cFN from the blood of patients with collagen disease presenting with abnormal elevations of cFN. The recent study by the inventors of the present invention uncovered that when the plasma of a patient with rheumatoid arthritis is subjected to cryofiltration, cFN is partially removed from the plasma, resulting in relief of the symptoms. However, the cryofiltration procedure is disadvantageous in that it involves discarding of the useful plasma protein in large quantities and is not capable of selective elimination of cFN. Thus, both a technology for selective elimination of cFN from the patient's blood nor a selective adsorbent for such elimination remained to be developed. Furthermore, detailed research into the physiological role of cFN in patients with collagen disease and the functions and physiological implications of pFN and cFN require pFN and cFN in the pure form but since the difference between pFN and cFN, in structure, is of the minuscule order on a molecular scale, they cannot be successfully fractionated from each other by known purification procedures [Katayama, M., J. Medical Technology, 34, 1725 (1990)]. For example, gelatin affinity chromatography [Hynes, R. O. (ed): Fibronectins, p9-14, Springer-Verlag, 1990] has heretofore been employed for the purification of FN but since there is no difference in the affinity for gelatin between pFN and cFN, even this method is not able to fractionate them from each other. Furthermore, because the molecular weight differential between pFN and cFN is extremely small, the routine purification methods for proteins, such as gel permeation chromatography, are also ineffective in the fractionation of these substances.
As the result of their intensive research, the inventors of the present invention developed a monoclonal antibody which recognizes the amino acid sequence of the EDA region of FN and using this antibody, successfully established a high-precision cFN assay method. Further research with the aid of this assay method led to the discovery that sulfated polysaccharides, represented by heparin, have a specific affinity for cFN. The inventors further made explorations starting from the above discovery and found that a water-insoluble crosslinked polysaccharide sulfate obtainable by subjecting the corresponding sulfated polysaccharide to crosslinking reaction and an immobilized polysaccharide sulfate obtainable by immobilizing a sulfated polysaccharide as a ligand on a water-insoluble support or carrier exhibit dissimilar adsorbent affinities for cFN and pFN and, hence, is a useful selective adsorbent for cFN. Based on these facts, the inventors developed a new technology by which pFN and cFN can be successfully fractionated from each other. The present invention has been developed on the basis of the above technology.